Arsenic Chemistry and Toxicity
Arsenic is a major contaminant of soils, sediments, wastes, and water in the United States and in foreign countries. Contamination of soils results from, for example, pesticides application and pressure-treated woods. Not only is arsenic a prevalent contaminant but it is also particularly dangerous because it is a known carcinogen. Currently there is no cost effective and efficient way to clean up sites contaminated with arsenic.
The use of arsenic in agricultural and industrial processes has resulted in numerous contaminated sites in Florida. During the early part of the 20th century, arsenic was commonly used as an insecticide component to control disease-carrying ticks on southern cattle so that Florida cattlemen could sell to the northern cattle markets. Arsenic, typically in the form of arsenic pentoxide, was also used in conjunction with copper sulfate and sodium or potassium dichromate as a wood preservative which is known as the copper/chromium/arsenic wood preservative process (CCA). With both of these processes, the risk of soil contamination from spills and leaks was great. The arsenic level at many of these sites is currently higher than 600 mg/kg even after years of idleness. The typical concentration range in soil is between 0.1 to 40 mg/kg, with a mean concentration of 5–6 mg/kg. The typical range of arsenic in Florida soils is 0.01 to 50.6 mg/kg.
In most soil systems, arsenic is present in many forms of which arsenate is typically the dominant one. In this form, it has properties very similar to phosphate including the formation of insoluble salts with cations and sorption by soil constituents. Because arsenic has a wide range of oxidation states (−3, 0, +3, and +5) it has the ability to form many types of organic and inorganic complexes. At high pH ranges, typically 7 to 9, the arsenic in soils predominantly consists of complex oxyanions of As(V), such as AsO2−1, AsO4−3, HAsO4−2, and H2AsO4−1. In soils with low pH and low Eh, the predominant forms of arsenic are the arsenite (H3AsO3).
Although arsenic is commonly found in all natural systems at minute levels, it can be very toxic to both plants and animals at higher concentrations. The toxic effects of arsenic have been known for some time. The exposure of animals to arsenic is second in toxicity only to lead (Pb) for many farm and household animals. Most cases of arsenic poisoning in animals occur in bovine and feline species as a result of contaminated feed supplies. Other species that are affected are forage-eating animals, such as horses and sheep, that encounter fields that may have been treated with arsenic pesticides. The toxic effects of arsenic to humans and animals can be related to the interactions that occur within the cells of poisoned individuals, especially the mitochondria.
Phytoextraction
Arsenic contamination in the environment is of concern due to its biological activities as a teratogen, carcinogen, and mutagen as well as its detrimental effects on the immune system. Due to the concern expressed over arsenic contaminated sites, various remediation techniques have been developed. Methods for remediating arsenic contaminated sites can be performed in situ and ex situ and have varying degrees of complexity, effectiveness, and cost. Due to the lack of effective technologies and the costs associated with the excavation and landfilling of the soil materials efforts to remediate these arsenic contaminated sites have been minimal. These remediation methods can be divided into three groups: chemical, physical, and biological remediation methods.
One of the biological remediation techniques is phytoremediation, more specifically phytoextraction. Phytoextraction attempts to remove contaminants from the rhizosphere through plant uptake and the contaminants are accumulated in roots, leaves and/or stems. The plant materials are then harvested and the contaminants reclaimed from the plant biomass or the materials are disposed of at a hazardous waste facility. Phytoextraction is an organic, low input, and solar energy powered remediation technique that is applicable to sites with surface and low to medium levels of contamination. The ideal plant for phytoextraction must be able to tolerate high levels of the element in root and shoot cells. Plants used for phytoextraction must have the ability to translocate the contaminant from roots to shoots at high rates. For most plants, root concentrations are much higher than shoot concentrations, but in hyperaccumulators, shoot metal concentrations exceed root.
There have been several reports of arsenic accumulating plants; on mine wastes from various sites in the United Kingdom; on smelter wastes in northeast Portugal and near a copper mine site in northern Peru. Porter and Peterson (1975) reported that Jasione montana, Calluna vulgaris, Agrostis tenuis and Agrostis stolonifera collected from highly arsenic polluted sites in the UK contained 6640, 4130, 3470 and 1350 μg As g−1 dry mass, respectively. De Koe (1994) found Agrostis castellana from the gold mines in Portugal reached arsenic values of 1900 mg kg−1 but was still in the range reported by Porter and Peterson (1975) for other Agrostis species. The highest As concentration previously reported in plants was for the grass Paspalum racemosum, which contained up to 5,280 μg As g−1 in their dead leaves.
Currently, many plants have been identified that can be utilized to remediate soil and water systems contaminated with metals, metalloids, petroleum constituents, pesticides, and industrial wastes. Also, many plant species have been identified that accumulate lead, selenium, nickel, zinc, and other metals. For example, U.S. Pat. Nos. 5,364,451 and 5,711,784 describe phytoremediation of metal-contaminated soils. For the remediation of contaminated sites contaminated with metals, phytoextraction can be an attractive option. Phytoextraction is the process of removing a contaminant from a system via plant roots for remediation purposes.
Pteris vittata 
There are more than 400 hyperaccumulators identified in different taxa mostly belong to nickel, cadmium, and zinc (Brooks, 1998). Recently, Ma et al. (2001) discovered the first known vascular plant, a fern, (Pteris vittata L.), commonly known as Chinese brake fern that hyperaccumulates arsenic. Pteris vittata took up phenomenal concentrations of arsenic (as high as 2.3%) from soil and allocated most of it to the aboveground fronds (up to 90%) for final storage (Tu and Ma, 2002). Most importantly, the hyperaccumulation of arsenic was accompanied by an increased biomass of the aboveground plant parts (Ma et al., 2001; Tu and Ma, 2002). Other desirable characters permitting P. vittata as an ideal plant for phytoremediation include its perennial growth habit, disease and pest resistance, fast vigorous growth, and diverse ecological niche with high pH.
Arsenic hyperaccumulation largely depends on the root geometry and morphology since root systems that have higher ratios of surface area to volume will more effectively explore a larger volume of soil. Pteris vittata develops an extensive network of root system enriched with root hairs. Bondada and Ma (2002) reported the root length and density of the fern grown in arsenic contaminated soil were 363 μm and 9 μm−2, substantially greater than the length and density of P. vittata grown in cadmium contaminated media (Gupta and Devi, 1994) indicating that arsenic may have stimulatory effect of root hair development in the fern. Since hyperaccumulation of metals appears to be driven by increased rates of root uptake, the dense population of root hairs in the fern, in addition to increasing absorptive surface, may contribute to increased rates of arsenic uptake by increasing number of transporters per gram fresh weight. Even though significant progress has been made in understanding the physiological basis of plant tolerance to arsenic, there remains considerable uncertainty about the mechanism in P. vittata. Tu et al. (2002) reported that P. vittata roots with low arsenic concentration and high phosphorus: arsenic ratio exhibited increased affinity to, and high influx rate of arsenic.
Pteris vittata has the remarkable ability to hyperaccumulate arsenic in the fronds, with frond concentrations reaching levels up to 100 fold greater than soil concentrations. This ratio is held both for uncontaminated (6 mg kg−1 As) and highly contaminated (1,500 mg kg−1 As) soils. The fern is capable of taking up of a range of inorganic and organic arsenic species including arsenate, arsenite and MMA. In the fern, arsenic is mostly present in inorganic forms, with 47–80% of the arsenic present as arsenite in the fronds.
Arsenic Uptake Other than Roots
Studies dealing with uptake of heavy metals by hyperaccumulators focused primarily on metal uptake from the soil solution via the root system. This is because most of the heavy metals reside in the soil system, and after uptake, they are often confined in the roots. Other than the roots, the aerial organs such as leaves are also capable of absorbing soluble heavy metals if they receive it in aqueous form (Lepp, 1975). Metals such as Cd, Zn, Cu, and Pb enter the leaf through foliar pathways, however, their entry through the leaf cuticle into leaf varied depending upon metal species (Little and Martin, 1972; Greger et al., 1993). Arsenic, a highly soluble metalloid, is normally applied in combination with other compounds as a toxin for pest mortality (Handson, 1984). In the past, however, foliar sprays of arsenic had been used to improve juice quality in citrus (Procopiou and Wallace, 1979) indicating that arsenic could gain entry into the plants through the foliar pathways.
Since different biochemical reactions occur in different parts of a plant, excised plant, such as shoots, stems, leaves and roots, have been widely used to characterize the absorption and metabolism of nutrients and chemicals as well as heavy metals in plants (Facanha and Okorokova-Facanha, 2002; Waldrop et al., 1996; Zhang and Taylor, 1991). We have examined the uptake of different As species (organic/inorganic and arsenate/arsenite) by P. vittata and As speciation in its plant biomass (Ma et al., 2001; Tu and Ma, 2002). However, there are many questions remain unanswered, such as where As reduction occurs in the plant, i.e. roots, fronds or both, and how P affects plant As uptake and reduction. The hypotheses were that both P and As species could affect plant As uptake, speciation and thiol formation in P. vittata, and such effects could be effectively characterized by use of excised parts of P. vittata. It was expected that use of excised parts of P. vittata to characterize As uptake, speciation and thiol formation would shed light on its mechanisms of As hyperaccumulation.
Although live biological systems work well for low concentrations, they cannot survive the high levels that are found in heavily contaminated areas and industrial effluents. The use of non-living biomaterial containing metal-binding compounds would have the advantage of not requiring care and maintenance as well as being useful in remediating areas with high levels of contaminants that would otherwise kill live systems. A wide variety of biomass, including bacteria, fungi, algae and higher plants have been tested as adsorbents to clean up metals in contaminated aqueous environments. Live or dead cultured cells of a higher plant, Datura innoxia Mill have been used to remove Ba2+ from solution. Aquatic ferns, Azolla filiculoides Lam and Azolla pinnata R.Br have also been reported to accumulate metals and can be used as biosorbents in remediating industrial effluents. A large number of aquatic plants were reported to be utilized for water purification and removing heavy metals from water. However, in aquatic plants, characterized by small size and slow growing roots, the efficiency of metal removal seems to be low. High water content in these plants renders their drying, composting and incineration processes complicated.
Terrestrial plants develop longer, fibrous root systems with root hairs, which creates a high surface area for effective absorption, concentration or precipitation of toxic metals from polluted media. An assessment of removal of toxic metals from solution by phytomass of Quercus ilex for a wide range of metals such as Cr, Ni, Cu, Cd and Pb indicated high sorption capacity of the phytomass for Ni and potential use as a biosorpent agent in contaminated aqueous media.
Prior to the subject invention, there has been no plant species identified that can enrich large quantities of arsenic into its biomass from contaminated soils, with arsenic concentration in plant being much greater than that in the soil. Also, prior to the subject invention there has been no report of the use of fern plants in phytoremediation. In addition, prior to the subject invention there has been no report of fern-based phytoremediation using the following methods:, e.g. foliar application, excised plant parts and dry or fresh plant biomass.